The invention relates to methods of formulation of viscoelastic biopolymers following bulk manufacture. The biopolymers to which the invention may be applied include homopolysaccharides and heteropolysaccharides, particularly the category of heteropolysaccharides known as glycosaminoglycans. Glycosaminoglycans especially suitable for use in the present invention are hyaluronic acid (HA), chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin and heparan sulfate.
HA is a naturally occurring biopolymer consisting of repeating disaccharide units of D-glucuronic acid in β-(1-3) linkage with N-acetyl-D-glucosamine, wherein each disaccharide unit is connected to its adjoining neighbors by β-(1-4) linkages. The salt sodium hyaluronate (NaHA) is found at physiological pH in human and vertebrate joint synovial fluid, connective tissue, vitreous humor of the eye and healthy skin tissue, and is an extracellular secretion product of several bacterial species, particularly of the genus Streptococcus. 
To date, the major medical applications of manufactured NaHA products are in ophthalmic surgery for cataracts and intraocular lens implantation, in dermatological applications for filling wrinkles and augmenting lip size, and in viscoelastic supplementation for the treatment of osteoarthritis in humans and large mammals. Viscoelastic supplementation, particularly in the knee, is aimed at restoring the normal rheological homeostasis of the joint network and for providing immediate protection, lubrication, shock absorption, hydrodynamic resistance and a mechanochemical barrier against stress. Intra-articular injections of NaHA have been shown to improve function and mobility and decrease pain.
More recent medical applications of manufactured NaHA include medical device coatings, surgical adhesion prevention products, drug delivery vehicles, bone replacement materials and wound healing materials.
Marketed NaHA products include BioLon™, Biolon™ Prime, BioHy™ (all from Biotechnology General (Israel) Ltd.), Hyalart™ (Fidia), Synvisc™ Hyalan G-F 20 (Biomatrix), Healon™ (Pharmacia), BD Vise™ (Becton Dickinson) and Orthovisc™ (Anika Therapeutics).
For commercial applications in the pharmaceutical, cosmetic and food industries, the quality of a viscoelastic biopolymer is dependent on the combined parameters of viscosity, concentration and molecular weight. For example, HA for pharmaceutical use must be of high molecular weight to ensure sufficient water retention, yet the viscosity must be of reasonable order so as to enable ease of administration and manipulation, e.g., syringeability. NaHA found in biological sources such as rooster combs and culture broths of fermented Streptococcus strains, is often of very high molecular weight, i.e., >3×106 daltons. Procedures used for its extraction, purification and sterilization typically result however, in a final product in which the molecular weight is significantly reduced as compared to the native compound.
For example, lyophilization of NaHA following repeated extraction induces sublimation of water, the net result of which is shearing of high molecular weight molecules. Low pH techniques result in formation of cross-links, which break upon subsequent pH increase and contribute to shearing of NaHA.
Sterilization techniques, such as those employing dry or moist heat, liquid chemicals, ethylene oxide gas, UV radiation, electron bean radiation, gamma radiation, microwaves and ultrasound all result in breakage of linear molecules, and are thus unsuitable for a viscoelastic biopolymer product in which molecular weight is a key parameter for optimal product quality.
NaHA manufacture from biological sources is well known to those skilled in the art. A typical bulk purification process (of which there are many variations) involves repeated extraction, precipitation, absorption, centrifugation and/or filtration steps to remove contaminants. Procedures for isolation of NaHA from fermented Streptococcus cultures are disclosed for example, in U.S. Pat. No. 4,780,414 and U.S. Pat. No. 4,784,990 (both assigned to BioTechnology General (Israel) Ltd.), U.S. Pat. No. 5,563,051, U.S. Pat. No. 5,411,874 (Fermentech Medical), U.S. Pat. No. 5,071,751 (assignee Chisso Corp.) and U.S. Pat. No. 5,316,916.
U.S. Pat. No. 5,023,175 (assignee Kabushiki) relates to purification of cosmetic grade NaHA (MW 2.1×106) with dialysis ultrafiltration as the final step prior to freeze drying.
U.S. Publication No. 2002/0120132 relates to purification of NaHA (MW>7.5×105) involving a temperature controlled reactor and allegedly less ethanol than traditional methods. The purified NaHA is dried under vacuum or lyophilized.
Alternate methods for bulk manufacture of NaHA from Streptococcal cultures avoid extraction/precipitation steps, and rely primarily on sequential filtration techniques as disclosed for example, in GB 2,249,315 (assignee Chisso Corp.), WO 95/04132 (applicant Fidia Corp.) and U.S. Pat. No. 6,489,467 (assignee Chemedica).
None of the above disclose processes for formulating a highly purified bulk manufactured viscoelastic biopolymer such as NaHA into a final sterile product suitable for medicinal use.
U.S. Pat. No. 4,141,973 (assignee Biotrics) discloses a sterile HA produced by extensive purification of material from rooster combs and dissolution of the final product in sterile phosphate buffered saline. The formulation however, has measurable amounts of protein and other impurities.
U.S. Pat. No. 4,517,295 (assignee Diagnostic Inc.) discloses a Streptococcal NaHA product prepared by a process in which sterile-filtration is the terminal step. The disclosed method suffers from the disadvantage of producing low molecular weight NaHA (average MW 5.5×104 daltons).
U.S. Pat. No. 5,093,487 (assignee Mobay Corp.) and U.S. Pat. No. 5,316,926 (assignee Miles Inc.) disclose final filter-sterilization of a Streptococcal NaHA formulation (average MW 1-2×106 daltons), following a purification method comprising a mechanical winding technique. The purpose of the winding technique is to increase both molecular weight and viscosity, after which heat treatment or filtration is performed allegedly to reduce viscosity without affecting molecular weight. The winding method is disadvantageous for its unknown effects on the chemical composition of NaHA.
U.S. Pat. No. 4,782,046 (assignee Mobay Corp.) relates to preparing a final NaHA product (average MW generally less than 3.0×106 daltons) by filter sterilization and/or beta-propiolactone treatment prior to syringe filling. Beta-propiolactone treatment is disadvantageous as traces may remain in the preparation following hydrolization, and it may adversely affect the chemical structure of NaHA.
U.S. Pat. No. 5,079,236 (assignee Hyal Pharmaceutical Corp.) relates to preparing a formulation by dissolving purified NaHA (average MW 5-20×104), optionally containing a steroid, in a heated solution of preservatives, e.g., sodium benzoate, methylparaben and propylparaben, adjusting the pH and volume, filling vials and sterilizing the vials in an autoclave. Autoclave sterilization is deleterious to NaHA molecular weight.
U.S. Pat. No. 5,411,874 and U.S. Pat. No. 5,563,051 (assignee Fermentech) relate to preparing a medical grade NaHA solution by dissolving NaHA (average MW 1.6-2.5×106) purified by repeated precipitations in sterile phosphate buffered saline.
WO 01/28602 (applicants Genetics Institute and Fidia) relates to injectable formulations comprising HA esters and osteogenic protein.
U.S. Pat. No. 6,221,854 (assignee Orquest) relates to injection formulations comprising NaHA and growth factors.
None of the above disclose industrially applicable methods for formulating bulk manufactured and purified viscoelastic biopolymer such as NaHA into a final product suitable for medicinal use.
An object of the invention is to provide a method for formulating a bulk manufactured and purified viscoelastic biopolymer such as NaHA into a final product suitable for medicinal use.
An object of the invention is to produce a formulation comprising high molecular weight viscoelastic HA that is suitable for administration by injection into ocular and intra-articular spaces in humans and animals.
An object of the invention is to provide an industrial process for formulating NaHA obtained by bulk manufacture into a final product of average molecular weight 3×106 or greater suitable for medicinal use without subjecting the NaHA to freeze-drying at any stage of manufacture or formulation.